Detector and screening device for ion channels

ABSTRACT

The invention provides for a detector assembly, fiber assembly and screening system for optical measurements.

FIELD OF THE INVENTION

[0001] The present invention generally relates to devices and methodsfor rapidly identifying chemicals with biological activity in liquidsamples, particularly automated screening of low volume samples for newmedicines, agrochemicals, or cosmetics.

BACKGROUND

[0002] Drug discovery is a highly time dependent and critical process inwhich significant improvements in methodology can dramatically improvethe pace at which a useful chemical becomes a validated lead, andultimately forms the basis for the development of a drug. In many casesthe eventual value of a useful drug is set by the timing of its arrivalinto the market place, and the length of time the drug enjoys as anexclusive treatment for a specific ailment.

[0003] A major challenge for major pharmaceutical companies is toimprove the speed and efficiency of this process while at the same timemaintaining costs to an absolute minimum. One solution to this problemhas been to develop high throughput screening systems that enable therapid analysis of many thousands of chemical compounds per 24 hours. Toreduce the otherwise prohibitive costs of screening such large numbersof compounds, typically these systems use miniaturized assay systemsthat dramatically reduce reagent costs, and improve productivity. Toefficiently handle large numbers of miniaturized assays it is necessaryto implement automatic robotically controlled analysis systems that canprovide reliable reagent addition and manipulations. Preferably thesesystems and the invention herein are capable of interacting in acoordinated fashion with other systems sub-components, such as thecentral compound store to enable rapid and efficient processing ofsamples.

[0004] Miniaturized high throughput screening systems require robust,reliable and reproducible methods of analysis that are sensitive enoughto work with small sample sizes. While there are a large number ofpotential analysis methods that can successfully used in macroscopicanalysis, many of these procedures are not easily miniaturizable, orlack sufficient sensitivity when miniaturized. This is typically truebecause absolute signal intensity from a given sample decreases as afunction of the size of the sample, whereas background optical ordetector noise remains more or less constant for large or small samples.Preferred assays for miniaturized high throughput screening assays havea high signal to noise ratios at very low sample sizes.

[0005] Fluorescence based measurements have high sensitivity and performwell with small samples, where factors such as inner filtering ofexcitation and emission light are reduced. Fluorescence thereforeexhibit good signal to noise ratios even with small sample sizes. Aparticularly preferred method of using fluorescence based signaldetection is to generate a fluorescent (emission) signal thatsimultaneously changes at two or more wavelengths. A ratio can becalculated based on the emission light intensity at the first wavelengthdivided by the emitted light intensity at a second wavelength. This useof this ratio to measure a fluorescent assay has several importantadvantages over other non-ratiometric types of analysis. Firstly theratio is largely independent on the actual concentration of thefluorescent dye that is emitting fluorescence. Secondly the ratio islargely independent on the intensity of light with which the fluorescentcompound is being excited. Thirdly the ratio is largely independent ofchanges in the sensitivity of the detector, provided that is that thesechanges are the same for the detection efficiency at both wavelengths.This combination of advantages make fluorescence based ratiometricassays highly attractive for high throughput screening systems, whereday to day, and, assay to assay reproducibility are important.

[0006] Fluorescence assays that produce ratiometric emission readoutshave gained in popularity as the advantages of the method have grown inacceptance. Changes in emission ratios at two more wavelengths can becreated through a number of distinct mechanisms including electronic andconformational changes in a fluorescence compound. Typically, thesechanges can occur in response to a chemical reaction or binding of thefluorescent compound to a particular ion such as a metal ion likecalcium or magnesium, or through a change in pH that influences theprotonation state of the fluorescent compound.

[0007] Alternatively ratiometric changes in emission can be convenientlybe obtained by exploiting the use of fluorescence resonance energytransfer (FRET) from one fluorescent species to another fluorescentspecies. This approach is predictable, sensitive and can give rise tolarge ratio changes at two well-defined and well spectrally resolvedwavelengths. Furthermore FRET can be generally applied to createratiometric assays for a range of activities. For example patent WO96/30540 (Tsien) describes a FRET based system to measure geneexpression using a fluorogenic substrate of beta lactamase. Patent WO96/41166 (Tsien) describes the use of a FRET based system to measurevoltage across the plasma membrane of a cell. Patent WO 97/20261 (Tsien)describes the use of FRET between two fluorescent proteins to measureintracellular protein. Such assays can be used with the inventionsdescribed herein.

[0008] The present invention is directed towards the development ofimproved optical systems for simultaneously measuring emission ratiosfrom a plurality of samples with high sensitivity, speed,reproducibility and accuracy. The present invention has severalimportant advantages over prior devices adapted to measure fluorescenceemission sequentially from samples.

[0009] Firstly, the simultaneous measurement of emission ratios enablesrapid fluctuations in lamp intensity, bleaching of the fluorescent dye,or cycle to cycle errors in the alignment of multiwell plates to becorrected for, thereby enabling much smaller changes in ratio to bereliably observed. Secondly, no mechanical movements are necessaryduring ratio measurement, eliminating mechanical design challenges.Thirdly ratios can be acquired very rapidly, as required for dynamicmeasurements of membrane potential or calcium, and are not limited bythe speed of filter changing. Fourthly the overall throughput and dutycycle are improved by eliminating dead times for filter changeover.Finally, residual ratio non-uniformities between addressable wellsshould be constant and easily correctable by using emission ratiospreviously measured on reference samples to normalize sample ratios insoftware.

SUMMARY OF THE INVENTION

[0010] The invention includes a method of simultaneously measuring atleast two optical properties of emitted light from at least one samplein a plurality of addressable wells of a multiwell plate comprising thesteps of,

[0011] i) aligning a plurality of addressable wells of a multiwell plateto a plurality of ball lenses;

[0012] ii) directing electromagnetic radiation substantially coaxiallythrough the symmetry axis of each of said plurality of ball lenses,

[0013] iii) detecting the emitted light focused by said plurality ofball lenses from said at least one sample.

[0014] The invention includes an optical detection system, comprising alight source that launches at least one predetermined wavelength oflight, sample holder, a ball lens at a predetermined interrogationdistance from said sample holder, a trifurcated fiber adapted for dualoptical interrogation and in optical communication with said ball lens,and a detector that detects light of at least one desired wavelength andin optical communication with said ball lens. Typically, the opticaldetection system includes a trifurcated fiber comprising a firstplurality of emission bundles for receiving light of a first wavelengthand second plurality of emission bundles for receiving light of a secondwavelength and said first plurality of emission bundles and said lightsource launches at least one predetermined wavelength of excitationlight at said sample holder. The optical detection system may furthercomprise at least one positioner to controllably change the spatialrelationship between the ball lens and the fiber or sample or acombination thereof. Typically, the light source launches light throughsaid trifurcated fiber to the location at least one addressable well ina sample in said sample holder to monitor epifluorescence. Preferably,the trifurcated fiber comprises an end that is generally at a focalplane of the ball lens.

[0015] The invention also includes an optical fiber assembly, comprisinga trifurcated fiber comprising a first plurality of emission bundles forreceiving light of a first wavelength and second plurality of emissionbundles for receiving light of a second wavelength and said firstplurality of emission bundles and said second plurality of emissionbundles are non-randomly distributed in plurality of excitation bundles.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 shows one embodiment of a fluorescence measuring deviceutilizing the detection system of the invention.

[0017]FIG. 2 shows one embodiment of a detection arrangement accordingto the invention.

[0018]FIG. 3 shows a cross sectional view of the fiber optic bundleshowing potential arrangements of the individual fiber optic fibers.Excitation fibers being represented by X or cross hatching, and emissionfibers being represented by the letters A for the first emission leg ofthe fiber optic bundle, and B, for the second emission leg fiber opticbundle.

[0019]FIG. 4 shows several embodiments of the ball lens of the inventionin a cross sectional view depicting the light directing ability of thelens.

[0020]FIG. 5 shows a perspective view of one embodiment of the ball lensassemblies of the present invention. The ball lens 500, ball lensholding assembly, 501 & 502 spring 503, fiber optic bundle 504, andmounting assembly for the assembly 505.

[0021]FIG. 6 shows a perspective view of one embodiment of the ball lensassembly Z-axis mover according to the invention. The stepper motor 600,z-axis mounting assembly 601, cam 602 & 603, ball lens assemblies 604,platform for ball lens assemblies 605, guiding pillar 606, switch 607,and trifurcated fiber optic bundle 608.

[0022]FIG. 7 shows a perspective view of one embodiment of a filterchanger of the invention. The filter holder enclosure 700 & 701, filterholder support 702 & 703, trifurcated fiber optic assembly 704,photomultiplier (PMT) 705, support 706, holding platform 707, and lighttight O-ring 708

[0023]FIG. 8A shows the rapid detection and continuous analysis ofvoltage changes induced within a cell measured using one preferredembodiment of the invention.

[0024]FIG. 8B shows a dose response curve of voltage changes inducedwithin a cell measured in response to the addition of an ion channelblocker, using one preferred embodiment of the invention.

[0025]FIG. 9 shows the use of one embodiment of a device comprising thetrifurcated ball lens assemblies of the invention to screen for ligandgated ion channel receptor antagonists.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

[0026] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention belongs. Generally, thenomenclature used herein and many of the automation, computer,detection, chemistry and laboratory procedures described below are thosewell known and commonly employed in the art. Standard techniques areusually used for engineering, robotics, optics, molecular biology,computer software and integration. Generally, chemical reactions, cellassays and enzymatic reactions are performed according to themanufacture's specifications where appropriate. The techniques andprocedures are generally performed according to conventional methods inthe art and various general references (see generally Lakowicz, J. R.Principles of fluorescence spectroscopy, New York: Plenum press (1983),and Lakowicz, J. R. Emerging applications offluorescence spectroscopy tocellular imaging: lifetime imaging, metal-ligand probes, multi-photonexcitation and light quenching. Scanning Microsc Suppl VOL. 10 (1996)pages. 213-24, for fluorescent techniques, Sambrook et al MolecularCloning: A laboratory manual, 2^(nd) ed. (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. for molecular biologymethods, Optics Guide 5 Melles Griot® Irvine, Calif. for general opticalmethods, Optical Waveguide Theory, Snyder & Love published by Chapman &Hall, and Fiber Optics Devices and Systems by Peter Cheo, published byPrentice-Hall for fiber optic theory and materials.

[0027] As employed throughout the disclosure, the following terms ,unless otherwise indicated, shall be understood to have the followingmeanings:

[0028] “Multiwell plate” refers to a two dimensional array ofaddressable wells located on a substantially flat surface. Multiwellplates may comprise any number of discrete addressable wells, andcomprise addressable wells of any width or depth. Common examples ofmultiwell plates include 96 well plates, 384 well plates and 3456 wellnanoplates.

[0029] “Addressable well” refers to spatially distinct location on amultiwell plate that may or may not have a physical representationoutside of the computer representation of the plate.

[0030] “Chemical plate” refers to a multiwell plate containingchemicals, such as stock solutions or dilutions thereof.

[0031] “Pharmaceutical agent or drug” refers to a chemical compound orcomposition capable of inducing a desired therapeutic effect whenproperly administered to a patient.

[0032] As used herein, “optical property” refers a measurable propertyof light, such as the intensity of emission light at a particularwavelength, the intensity or degree of light polarization, thetransmittance of a compound or composition, or the reflectance of acompound or composition.

[0033] “Ball lens” refers to a sphere, truncated sphere, cylinder, ortruncated cylinder of suitable transparent refractive material and isusually a sphere.

[0034] “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner.

DESCRIPTION OF ONE EMBODIMENT OF THE INVENTION

[0035]FIG. 1 shows one device of the invention. In one embodiment of theinvention, a device integrates a liquid handler 115, a multiwellpositioning stage 112 and a detection device comprising the ball lenstrifurcated plates containing cells and compounds are loaded into thedevice either manually or by a robotic system. The device then takes theplate(s) into the light-tight reading area 116. The equipment mayinclude a liquid handler 115 (such as a modified Hamilton Micro Lab 2200MPH, Hamilton Co, Reno, Nev.), and at least 1 dispensing tip 114, amulti-well plate positioning stage (500000 series, Parker Hannifin Corp,Harrison City, Pa.), in addition to the device of the invention. Basalfluorescence readings are made at both emission wavelengths prior tocompound addition.

[0036] In one embodiment the system was designed to simultaneouslymeasure fluorescence at two different emission wavelengths from a columnof 8 wells before, during, and after the introduction of a fluid sampleobtained from another multiwell plate or trough.

[0037] A compound or compounds are then added to the cells whilefluorescence at both emission wavelengths is continuously measured.After the whole plate is read, it is moved out of the light-tightenclosure and retrieved by the manual user or robotic system.

[0038] 16 photomultipliers are used to detect fluorescence emission at arate of 1 Hz or 10 Hz. Both exciation and emission wavelengths areselected via interference filters. The 300 W xenon arc lamp providesillumination from 350 nm to 650 nm. The multi-alkali photomultiplier(Hamamatsu HC124-01) tubes can detect wavelengths ranging from 300 to850 nm. The bi-alkali photomultiplier tubes can detect emissionwavelengths from 300 to 650 nm. Two photomultiplier tubes are used todetect fluorescence from each well in a column of 8 wells allowing forcontinuous emission ration detection. The blue-sensitive bi-alkaliphotomultiplier tube is typically used to detect the shorter wavelengthemission while the multi-alkali photomultiplier tube is used to detectlonger wavelength emission.

[0039] The vertical position of the fibers is adjusted by a steppermotor driven cam system. The fibers are lowered when the plate is movedin or out of the system to allow the skirt of the microplate to passover the fibers. The fibers are raised once the plate is in the systemto maximize fluorescence detection efficiency.

[0040] The microplate carrier is extended through a trap door 112 by astepper motor driven translation stage to receive microplates fromeither the robotic system or a manual operator.

[0041] The liquid handler is then triggered via the multifunction boardI/O feature to add reagent to the wells while fluorescence iscontinually read. The plate is then moved to the next column where thisprocess is repeated. Each column is read in this manner until the wholeplate has been read. The plate is then extended out of the instrumentand retrieved by either a robotic system or the manual operator. Thesystem is now ready to receive another plate. The device can be alsoconfigured to read in a different manner in which the whole plate isread at once rather than one column at a time. In the “plate assay mode”the whole plate is read a fixed number of times. The plate is thenstopped and the Hamilton is triggered to add reagents to the wholeplate. The plate is then read for an additional number of timesfollowing reagent addition. Upon completion of reading, the plate isonce again extended out of the system.

[0042] Fluorescence can be detected from an interrogation layer ifdesired using the invention. Referring to FIG. 2, monolayers of cellscan be detected on the bottom of microplate wells 206 by the common endof a trifurcated optical fiber bundle 203. One leg of the eachtrifurcated fiber bundle is used as an excitation source 201; each ofthe eight excitation legs is fused into a single bundle 204 to provideuniform light intensity to each of the eight trifurcated bundles. Theother two legs of the trifurcated fiber are used for to detectfluorescence emission 214 and 213. The common end of the trifurcatedbundle is used to both excite and collect fluorescence emission. 8trifurcated fibers are used to detect two emission channels from eachwell in a column of 8 wells. A ball lens 205, (RB-707004, BirdPrecision, Waltham, Mass.) is at the top of the common end of thetrifurcated fiber bundle to increase the efficiency of fluorescencedetection.

[0043] A 300 watt xenon arc lamp 201, CXP300, ILC Technology, Sunnyvale,Calif.) with a parabolic reflector can be used as the fluorescenceexcitation source. The excitation light is filtered by two 2″ diameterinterference filters (400RDF15 or 480RDF20, Omega Optical, Brattleboro,Vt.) and then focused by a lens 202 on to the excitation leg of thetrifurcated bundle. Both a IR heat absorbing water filter 207 andshutter system 208 are also included in the optical path to protect theinterference filters from heat damage. A 1″ diameter “head-on”photomultiplier (HC124 series, Hamamatsu Corp, Bridgewater, N.J.) tubesare used to detect the fluorescence emission. Fluorescence emission fromone leg of the fiber bundle is detected by a blue-sensitive bi-alkalaiphotomultiplier tube 209; emission from the other leg of the fiberbundle is detected by a red-sensitive multi-alkalai photomultiplier tube210. Data is collected by the A/D portion of a multifunction board(PCI-MIO-E-1, National Instruments, Dallas, Tex.) in a pentium basedpersonal computer 212. The computer controls data acquisition, plate andfiber movement, and shutter opening and closing 215.

[0044] The device can utilize a Hamilton Mircolab 2000 8-channelautomated liquid handler to add reagents to the top of a 96 well plate.The Hamilton Eclipse software allows the user to change parameters suchas reagent volume, speed of reagent addition, height at which reagentsare added, and wash cycle length. Example programs are provided for theuser to customize for their assay. Separate positions are provided forthe 96 well assay plate and a 96 well plate containing compounds. Atypical assay where each column is read for 35 seconds takes about 7.3minutes per plate allowing for 30 plates to be read in less than 4hours. The fastest the system can be run using current liquid additionand washing protocols is 20 seconds per column or 4.3 minutes per plateallowing for 30 plates to be read in about 2.5 hours.

COMPONENTS OF THE DETECTION SYSTEM

[0045] Typically, the greatest issue in fluorescent detection is thereduction of background signal in the detection system. In this case thedetection system might comprise the excitation source and associatedoptics (dichroic filters, interference filters, focussing lenses,collimators etc), the fiber optic assembly (exicitation and emissionpathways and patterns), the substrate containing sample to be analyzed,and the emission filters and associated optics that direct the emissionradiation to the detection element. A key challenge in epifluorescentdetection (where the excitation light and emission light are directedand collected from the same plane) is to maximize the excitation lightenergy and the area (the field of view created by the excitation light)this energy is delivered to the sample without comprising the efficientcollection of the fluorescent emission or generating a high backgroundfrom the reflection of excitation radiation. Typically, a tradeoffexists between optimal radiant energy, the field of view illuminated bythe excitation energy and the fluorescent emission collectionefficiency. For example, the wavelength to be utilized for excitationmay preclude the use of certain materials (which might have otherdesirable features like high NA) due to the incompatibility of thematerial (high autofluorescence) with the excitation wavelength that isrequired.

[0046] Fiber optic bundles are created with varying packing patterns ofexcitation and emission bundle arrangements, and with different numbersof fibers in the excitation leg and dual emission legs. In oneembodiment, the packing of the fibers of both the excitation andemission legs in the bundle is randomly packed. In another embodimentthe fibers are arranged in specific and defined patterns, that confers apreferred optical characteristic to the system. For example, theexcitation fibers could be bundled to together centrally in the fiberoptic bundle and the emission filters arranged around the outside tocreate a coaxial fiber optic bundle. Alternatively, the emission bundlescould be arranged in small groups to create an array, or radially aroundthe axis of the bundle, or any other symmetrical or non-symmetricalpattern.

[0047] Fiber optic assembles may also vary in total number of fibers ofboth the excitation and emission legs and overall size. The number ofexcitation fibers and the number of emission fibers and the relativeratios of excitation fibers to emission fibers may be widely varieddepending upon the other components in the system, as well as the typeof light source, sensitivity of the detector and size of the addressablewell in which the sample is located. The optimization of these factorsis discussed herein. In one embodiment a fiber optic bundle may containa total of 341 fibers of which 55 will be excitation fibers arrangedrandomly within the fiber. In another embodiment the fiber may have 341fibers of which 85 fibers are excitation fibers arranged inpreferentially within the center of the bundle, but also distributedrandomly through the remainder of the emission bundles. In anotherembodiment the fiber may contain 112 fibers of which 7 fibers areexcitation fibers arranged in the center of the bundle, and theremaining emission fibers are located around the excitation fibers. Inanother embodiment the fiber may contain 1417 fibers of which 163 fibersare excitation fibers arranged in the center of the bundle, and theremaining emission fibers are located around the excitation fibers. Inanother embodiment of the fiber optic bundle, the excitation fibers arecentrally located within the bundle and extend beyond the point wherethe emission fibers terminate. In a preferred version of this embodimentthe emission filters terminate into a liquid light guide that is incontact with the ball lens.

[0048] Ball lens compositions of materials of different refractive indexand of different sizes can be easily evaluated with each fiber opticarrangement to establish a preferred optical arrangement. Ball lens ofabout 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 8 mm, 10 mm and 20 mm diametermay be evaluated depending on the size of the instrument and spatialrequirements of the imaging system desired. Suitable compositions of theball lens include fused silica, sapphire, optical glass (such as BK7,SF11 or LaSF9), borosilicate glass or zinc selenide (for infraredapplications). Preferred compositions of the ball lens for use withinthe wavelength range 300-750 nm include fused silica and sapphire. Forlow light applications it is often necessary to include a suitableanti-reflective coating such as single or multi-layer MgF₂, V-coatings,HEBBARTM™ (High Efficiency BroadBand AntiReflection) and Extended rangeAntiReflective coatings for a ball lens. To determine the optimumcomposition, size and AntiReflective coating (AR) coating of the lensdifferent coatings, each size of ball lens above made of each of thematerials above would be prepared with each of the AR coatings above,and in the absence of an AR coating.

[0049] To select the preferred optical components for a specificapplication it is often preferred to determine the signal to noise (S/N)level for particular combinations of ball lens and fiber opticassemblies. Signal to noise ratios can be determined by comparing themagnitude of a defined amount of fluorescent material measured in theoptical system, compared to the noise obtained by measuring an emptywell under exactly the same conditions. S/N ratios can be calculated ata range of concentrations of the calibration material (for example,fluorescein) to determine overall detector sensitivity and linearity.Additionally, variability of measurements can be expressed in terms ofstandard deviation (S.D.) and Coefficient of Variance (C.V.) toestablish reproducibility and alignment sensitivity of each of thesystems.

[0050] Additionally, it is preferred to select the spacing of the fiberoptic bundle to the ball lens and the ball lens to the surface of theobject to be interrogated. This can be quickly accomplished bygenerating a graph of S/N ratio versus distance for each of the opticalarrangements desired. In the same way, similar S/N ratio graphs can becreated for each of the combinations in response to differentillumination intensities and wavelengths of excitation light (inconjunction with appropriate fluorescent samples). This analysis wouldcreate a matrix of performance characteristics as represented by S/Nratios that are used to select the optimal fiber optic assemblies, balllens size, composition, antireflective coating, and spatial alignmentsof the components for specific applications.

[0051] Detectors

[0052] The detector can include a photon sensitive surface or materialfor measuring photon emission, such as a CCD, photodiode, or a PMT. Thedetector can intensify the signal, and gate if desired, using a photonintensifier. Preferably, the detector can utilize a high quantumefficiency CCD without an intensifier for long detection integration.Alternatively, the detector can utilize PMT's or multi-site PMT's forphoton detection and quantitation.

[0053] The detector preferably functions in the epi-fluorescence modewhere the preferred illumination is from the bottom of the multiwellplate and the preferred collection is also from the bottom of themultiwell plate. The detector usually is capable of three to four ordersof magnitude of dynamic range in signal response from a single reading.The detector, in a preferred embodiment, utilizes a CCD chip for imagingand detecting photons emitted from the assay wells.

[0054] Light Source

[0055] In the preferred embodiment, the detector comprises a lightsource assembly (e.g., Xenon) that can be switched between continuousand pulsed (1 kHz) output depending upon power supply. Suitable lightsources are described herein and other suitable sources can be developedin the future.

[0056] Liquid Handlers

[0057] In one embodiment, the liquid handler can comprise a plurality ofnanoliter pipetting tips that can individually dispense a predeterminedvolume. Typically, pipetting tips are arranged in two-dimension array tohandle plates of different well densities (e.g., 96, 384, 864 and3,456).

[0058] Usually, the dispensed volume will be less than approximately2,000 nanoliters of liquid that has been aspirated from a predeterminedselection of addressable wells and dispensed into a predeterminedselection of addressable wells. Preferably, nanoliter pipetting tips candispense less than approximately 500 nanoliters, more preferably lessthan approximately 100 nanoliters, and most preferably less thanapproximately 25 nanoliters. Dispensing below 25 nanoliters can beaccomplished by pipetting tips described herein. Preferred, minimalvolumes dispensed are 5 nanoliters, 500 picoliters, 100 picoliters, 10picoliters. It is understood that pipetting tips capable of dispensingsuch minimal volumes are also capable of dispensing greater volumes. Themaximal volume dispensed will be largely dependent on the dispense time,reservoir size, tip diameter and pipetting tip type. Maximum volumesdispensed are about 10.0 microliters, 1.0 microliters, and 200nanoliters. Preferably, such liquid handlers will be capable of bothdispensing and aspirating. Usually, a nanoliter pipetting tip (orsmaller volume dispenser) comprises a fluid channel to aspirate liquidfrom a predetermined selection of addressable wells (e.g., chemicalwells containing drug candidates). Liquid handlers are further describedherein, and for some volumes, typically in the microliter range,suitable liquid pipetting tips known in the art or developed in thefuture can be used. It will be particularly useful to use liquidhandlers capable of handling about 1 to 20 microliter volumes when it isdesired to make daughter plates from master plates. Preferably, in suchinstances a liquid handler has a dispensing nozzle that is adapted fordispensing small volumes and can secure a tip having a fluid reservoir.

[0059] In one embodiment nanoliter pipetting tips comprise solenoidvalves fluidly connected to a reservoir for liquid from an addressablechemical well. The fluid reservoir can be a region of a dispenser thatcan hold fluid aspirated by the nanoliter pipetting tip. Usually, a tipreservoir will hold at least about 100 times the minimal dispensationvolume to about 10,000 times the dispensation volume and more preferablyabout 250,000 times the dispensation volume. The solenoid valves controla positive hydraulic pressure in the reservoir and allow the release ofliquid when actuated. A positive pressure for dispensation can begenerated by a hydraulic or pneumatic means, e.g., a piston driven by amotor or gas bottle. A negative pressure for aspiration can be createdby a vacuum means (e.g., withdrawal of a piston by a motor). For greaterdispensing control, two solenoid valves or more can be used where thevalves are in series and fluid communication.

[0060] In another embodiment, nanoliter pipetting tips comprise anelectrically sensitive volume displacement unit in fluid communicationto a fluid reservoir. Typically, the fluid reservoir holds liquidaspirated from an addressable chemical well. Electrically sensitivevolume displacement units are comprised of materials that respond to anelectrical current by changing volume. Typically, such materials can bepiezo materials suitably configured to respond to an electric current.The electrically sensitive volume displacement unit is in vibrationalcommunication with a dispensing nozzle so that vibration ejects apredetermined volume from the nozzle. Preferably, piezo materials areused in dispensers for volumes less than about 10 to 1 nanoliter, andare capable of dispensing minimal volumes of 500 to 1 picoliter. Piezopipetting tips can be obtained from Packard Instrument Company,Connecticut, USA (e.g., an accessory for the MultiProbe 104). Suchdevices can also be used in other liquid handling components describedherein depending on the application. Such small dispensation volumespermit greater dilution, conserve and reduce liquid handling times.

[0061] In some embodiments, the liquid handler can accommodate bulkdispensation (e.g., for washing). By connecting a bulk dispensationmeans to the liquid handler, a large volume of a particular solution tobe dispensed many times. Such bulk dispensation means are known in theart and can be developed in the future.

[0062] Positioners, Transitional Stages

[0063] Interrogation, aspiration or dispensation into multiwell platesof different densities can be accomplished by automated positioning(e.g. orthogonal) of a multiwell plate. Typically, the multiwell platesare securely disposed on an orthogonal positioner that moves the wellsof a multiwell plate with a first density in an X,Y position withrespect to the X,Y position of the liquid handler. Usually, the liquidhandler will have an array of aspiration and/or dispensation heads, orboth. Many aspiration/dispensation heads can operate simultaneously. Theorthogonal positioner will align each addressable well with theappropriate dispensing head. Preferably, a predetermined location (e.g.,center) of a pre-selected addressable well will be aligned with thecenter of a dispensing head's fluid trajectory. Other alignments can beused, such as those described in the examples. With a head substantiallysmaller than a well diameter, orthogonal positioning permits aspirationor dispensation into plates of different densities and well diameters.

[0064] An orthogonal positioner can typically match an array ofdispensing heads with an array of addressable wells in X,Y using amechanical means to move the addressable wells into position or theliquid handler (e.g., dispensing heads) into position. Preferably,arrays of addressable wells on a plate are moved rather than the liquidhandler. This design often improves reliability, since multiwell platesare usually not as heavy or cumbersome as liquid handlers, which resultsin less mechanical stress on the orthogonal positioner and greatermovement precision. It also promotes faster liquid processing timesbecause the relatively lighter and smaller multiwell plates can be movedmore quickly and precisely than a large component. The mechanical meanscan be a first computer-controlled servo motor that drives a basedisposed on a X track and a second computer-controlled servo motor thatdrives a Y track disposed on the X track. The base can securely disposea multiwell plate and either a feedback mechanism or an accurateCartesian mapping system, or both that can be used to properly alignaddressable wells with heads. Other such devices, as described herein,known in the art or developed in the future to accomplish such tasks canbe used. Usually, such devices will have an X,Y location accuracy andprecision of at least ±0.3 mm in X and ±0.3 mm in Y, preferably of atleast ±0.09 mm in X and ±0.09 mm in Y, and more preferably of at least±0.01 mm in X and ±0.01 mm in Y. It is desirable that such devicescomprise detectors to identify the addressable wells or multiwell platesbeing orthogonally positioned. Such positioners for predetermined X, Ycoordinates can be made using lead screws having an accurate and finepitch with stepper motors (e.g., Compumotor Stages from Parker, RohnertPark, Calif., USA). Positioners (eg. X, Y or Z) can be used to move thedetector assembly, the sample, liquid handler or a combination there of.

[0065] Alternatively, the liquid handler can be disposed on aZ-positioner, having an X,Y positioner for the liquid handler in orderto enable precise X,Y and Z positioning of the liquid handler (e.g.,Linear Drives of United Kingdom).

[0066] A reference point or points (e.g., fiducials) can be included inthe set up to ensure that a desired addressable well is properly matchedwith a desired addressable head. For instance, the multiwell plate, theorthogonal positioner or the liquid handler can include a referencepoint(s) to guide the X,Y alignment of a plate, and its addressablewells, with respect to the liquid handler. For example, the liquidhandler has a detector that corresponds in X,Y to each corner of aplate. The plate has orifices (or marks) that correspond in X,Y to theliquid handler's position detectors. The plate's orifices allow light topass or reflect from a computer-controlled identification light sourcelocated on the orthogonal positioner in the corresponding X,Y position.Optical locators known in the art can also be used in some embodiments(PCT patent application WO91/17445 (Kureshy)). Detection of light by theliquid handler emitted by the orthogonal positioner verifies thealignment of the plates. Once plate alignment is verified, aspiration ordispensation can be triggered to begin. Stepper motors can be controlledfor some applications as described in U.S. Pat. No. 5,206,568(Bjornson).

[0067] The liquid handler will also typically be disposed on aZ-dimensional positioner to permit adjustments in liquid transferheight. This feature allows for a large range of plate heights andaspirate and dispense tips, if desired, to be used in the sampledistribution module. It also permits the dispense distance between aaddressable well surface, or liquid surface in an addressable well, anda liquid handler to be adjusted to minimize the affects of staticelectricity, gravity, air currents and to improve the X,Y precision ofdispensation in applications where dispensation of a liquid to aparticular location in a addressable well is desired. Alternatively,multiwell plates can be positioned on a Z-dimensional positioner topermit adjustments in liquid transfer height. Static neutralizingdevices can also be used to minimize static electricity. Generally, theliquid transfer height will be less than about 2 cm. Preferably, smallvolumes will be dispensed at a liquid transfer height of less than about10 mm, and more preferably less than about 2 mm. Occasionally, it may bedesirable to contact the tips with a solution in a controllable fashion,as described herein or known in the art

[0068] Control, Data Processing and/or Integration Modules

[0069] In one embodiment, a data processing and integration module canintegrate and programmably control a liquid handler module, a movingconveying surface, and a detector module to facilitate rapid processingof the multiwell wells. To manage information in the system, the dataprocessing and integration module comprises elements to store, manageand retrieve data, including a data storage device and a processor. Thedata storage device can hold a relational database, an array of physicaldisk drives (e.g., random access disk drives), and a connection to othersystem components via a network. A data storage device can, forinstance, store a relational database for environmental, diagnostic, anddrug discovery applications. For instance, one particularly usefulrelational database can be provided by Oracle, and the network can be aTCP/IP (transfer communication protocol) ethernet LAN (local areanetwork).

[0070] Software Controls

[0071] The system can be controlled using supervisory control programs,which are not necessarily located on the same computer as the datastorage device. For example, in one embodiment of a system, a separatesupervisory control computer is provided for each of the Storage andRetrieval, Reagent Transport, and Reagent Distribution functions. Asupervisory control computer is a computer programmed to control aparticular subsystem using data from the data storage device andoperating on a workstation or component, such as a storage andretrieval, reaction module or sample transporter. Within the datastorage device, exists a structure for information in the form of tablesand relations. This structure is designed to meet the specific needs ofthe system, wherein it must accommodate the throughput demands of anautomated system and facilitate the presentation of information foranalysis and visualization of results. The data storage device cantypically process in excess of 100,000 transactions (read or writeparticular data) per day, while accurately keeping track of everychemical, biological reagent, operation, unit of work and workstationand other related activities. Integrity of the data storage device istypically maintained for simultaneous multiple users and processes.Information in the relational database of the data storage device isused to define operations to be performed, and a complete audit trailcan be maintained of every operation on every unit of work throughoutthe system.

[0072] Storage devices suitable for use with the present invention arewell known and are commercially available from a number ofmanufacturers, such as the 2 gigabyte Differential System Disk, partnumber FTO-SD8-2NC, and the 10 gigabyte DLT tape drive, part numberP-W-DLT, both made by Silicon Graphics, Inc., of Mountain View, Calif.,or equivalents (e.g., optical discs). A preferred embodiment usesHewlett Packard 4 GB Hot Swap Drives in a Netserver LX Pro configured asRAID-5

[0073] Interface Designs

[0074] In most embodiments, it will be advantageous to integrate andoperably link device of the invention with at least one otherworkstation, usually a sample transporter. The integration can beaccomplished with a computer and associated control programs to instructthe translational stage and sample processor to operate coordinately.Alternatively, the device may be used without directly integrating toanother workstation by tracking addressable wells in groups and eithermechanically or manually transporting multiwell plates to another workstation where the multiwell plates are identified. For instance, thedevice of the invention may be directly integrated and operably linkedto a storage and retrieval module and sample transporter, and indirectlylinked to a integration and control module. While this approach isfeasible, especially for lower throughputs, it is not desirable forhigher throughputs as it lacks direct integration that can lead tofaster throughput times. Manual operations also are more frequentlysubject to error especially when processing large numbers of samples.Preferably, the device of the invention can be integrated with otherworkstations and operate in a mode with minimal or substantially nomanual intervention related to transferring multiwell plates to otherwork stations.

[0075] Usage Modes

[0076] The detector is often capable of many different operating modesthat facilitate drug discovery assay requirements. These operating modescan include: single excitation wavelength with single emissionwavelength detection, single excitation wavelength, dual emissionwavelength detection, sequential dual excitation wavelength with dualemission wavelength detection and ratio measurement determination,sequential dual excitation wavelength with four emission wavelengthdetection and ratio measurement determination, homogeneous time resolvedfluorescence with single excitation wavelength and single emissionwavelength detection, homogeneous time resolved fluorescence with singleexcitation wavelength and dual emission wavelength detection and ratiodetermination measurement, homogeneous time resolved fluorescence withsequential dual excitation wavelength and dual emission wavelengthdetection and ratio determination measurement, absorbance (e.g. dual),transmittance (e.g. dual), reflectance, dual sequential excitationwavelengths and single emission wavelength detection with ratiodetermination measurement, luminescence measurement at a singlewavelength with luminescence measurement at dual wavelengths,luminescence measurement at dual wavelengths with a ratio determination,and time resolved fluorescence emission (intrinsic dye properties withor without a binding event).

[0077] Software and Data Collection

[0078] A windows based graphical user interface can be used with theinvention. The software calls routines, which will either setup theinstrument, test the instrument, run manual assays, run automatedassays, or analyze data. Users can change such parameters as how longdata is acquired, how compound additions are performed, and when in thetime trace the compound is added. A safety feature to check whether aplate has been loaded can also be set in the control screen. Manual testfunctions include moving the 96 well plate in and out, raising andlowering the optical fibers, and reading fluorescence values. Assays canbe run in two different formats as discussed in the hardware section.The experiments can be controlled by a Sagian robotic system or by amanual user. The user can also change liquid addition parameters such asvolume of reagent added or speed of liquid addition in the HamiltonMicrolab 2200 software.

[0079] Raw intensity values for both emission wavelengths can be plottedin real-time. Data can be analyzed and the result displayed immediatelyfollowing the completion of an experiment. Both raw and processed datafiles are saved as ASCII text. The data files can be imported byspreadsheet programs (e.g. Excel) for further analysis. Software andhardware have been provided to allow a robotic system to load and removeboth compounds and assays plates. Software to allow the user tooptionally introduce a manual bar code reader has also been included.

[0080] Fluorescence Measurements

[0081] It is recognized that different types of fluorescent monitoringsystems can be used to practice the invention with fluorescent probes,such as fluorescent dyes or substrates. Preferably, systems dedicated tohigh throughput screening, e.g., 96-well or greater microtiter plates,are used. Methods of performing assays on fluorescent materials are wellknown in the art and are described in, e.g., Lakowicz, J. R., Principlesof Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B.,Resonance Energy Transfer Microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed.Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.219-243; Turro, N. J., Modern Molecular Photochemistry, Menlo Park:Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361 and theMolecular Probes Catalog (1997), OR, USA.

[0082] Preferably, FRET (fluorescence resonance energy transfer) is usedas a way of monitoring probes in a sample (cellular or biochemical). Thedegree of FRET can be determined by any spectral or fluorescencelifetime characteristic of the excited construct, for example, bydetermining the intensity of the fluorescent signal from the donor, theintensity of fluorescent signal from the acceptor, the ratio of thefluorescence amplitudes near the acceptor's emission maxima to thefluorescence amplitudes near the donor's emission maximum, or theexcited state lifetime of the donor. For example, cleavage of the linkerincreases the intensity of fluorescence from the donor, decreases theintensity of fluorescence from the acceptor, decreases the ratio offluorescence amplitudes from the acceptor to that from the donor, andincreases the excited state lifetime of the donor. Preferably, changesin signal are determined as the ratio of fluorescence at two differentemission wavelengths, a process referred to as “ratioing.” Differencesin the absolute amount of probe (or substrate), cells, excitationintensity, and turbidity or other background absorbances betweenaddressable wells can affect the fluorescence signal. Therefore, theratio of the two emission intensities is a more robust and preferredmeasure of activity than emission intensity alone.

EXAMPLES Example 1 Construction and Testing of a Ball Lens TrifurcatedFiber Optic Assembly

[0083] Arrangements of ball lenses and trifurcated fibers can betailored to their intended application. To determine the appropriatearrangement of fiber optic bundles and ball lens a series of experimentscan be conducted to determine the highest signal to noise ratio,preferred sensitivity, lowest background, preferred field of opticalinterrogation or excitation or a combination thereof.

[0084] For example, one embodiment of a trifurcated fiber optic assemblyadapted for miniaturized sample analysis of a 1 mm well diameter with avariable interrogation layer of approximately 0.1 mm to 2.0 mm couldcomprise the following arrangement. A ball lens made of fused silicamaterial coated with an antireflective coating such as HEBBAR, with adiameter of about 3 mm. A trifuricated fiber optic assembly, opticallycoupled to the ball lens comprising 91 fibers of which 7 fibers in thecenter are for excitation and the remaining fibers are for emissioncollection. The fiber assembly being about 3 mm in diameter and packedinto a hexagonal ferrule to maximize packing efficiency and ease ofassembly. The emission fibers (41 for each optical property to bemeasured) are selected so as to maximize collection efficiency, signalintensities and signal to noise or signal to background) properties ofthe assembly. The following table illustrates the effect that thespatial position of the ball lens to the fiber assembly has onSignal-to-background. In this example, the distance of the ball lens tothe test fluorescent sample was kept constant as the distance of theball lens to the fiber optic assembly was varied. Different signal tobackground ratio's were obtained from which an optimal distance could beselected. TABLE 1 Emission filter: 535RDF30 (no long pass) Samplevolume: 2 microliters, hand loaded. Ball lenses: Fused silica. Distancefrom lens to plate = 0. (Signal − background)/ Fiber-to-lens EmptyBuffer (BB) 10 nM F background (mm) (mV) Background Signal (10 nM F −BB)/BB 0 10.8 12.4 119 9 0.384 9 7.95 117.67 14 0.482 7.8 6.95 130.33 180.533 8 7.25 126 16 0.584 7.9 7.15 122 16 0.71 4.9 5.8 34 5

[0085] Subsequent further analysis of this particular systemdemonstrates an optimal distance for the test sample relative to theball lens fiber optic assembly between about 0 and 0.152 mm. TABLE 2Distance from fiber to lens = 0.584 mm lens-to-plate Empty Buffer (BB)10 nM F (mm) (mV) (mV) (mV) (10 nM F − BB)/BB 0 7.9 7.15 122 16 0.1527.8 7.2 126 17 0.203 8.4 8.15 119.5 14 0.305 10 8.9 118 12

[0086] In one example a 3 mm Hebbar coated sapphire ball lens wasutilized with four different 665 fiber optic assemblies (as shown inFIG. 3) to assess performance as measured by minimum detectable level ofsignal for the particular dye being tested as described herein. TABLE 3Detectable Number Number signal of of Fiber Description in nM of Ex EmAssembly of Assembly Fluorescein Fibers Fibers Diameter Assembly #1 0.507.00 84.00 2.6 mm Assembly #2 0.86 1.00 6.00 1.2 mm Assembly #3 0.503.00 16.00 1.5 mm Assembly #4 0.50 7.00 30.00 1.6 mm

[0087] Surprisingly, assemblies #3 and #4 perform as well as assembly#1. This indicates that the optimal ball lens size can be typicallyabout equal or 1 to 3 times greater in diameter than the fiber opticassembly. The ball lens can thus aid in reducing the complexity orquantity of fibers required in a fiber optic assembly for optimaldetection sensitivity particularly when the need to reduce the size ofthe fiber optic assembly is important in a miniaturized system.

[0088] In a similar example to above, the fiber assembly is keptconstant but the size of the ball lens is varied. In this example a 3 mmdiameter coaxial fiber optic assembly containing 112 fibers arrangedwith 7 XXF200/210/235T Fused Silica excitation fibers in the center ofthe assembly surrounded by 105 XXF200/210/235T Fused Silica emissionfibers. 3 different size sapphire ball lenses are compared, a 3 mm lens,a 5 mm lens and a 10 mm lens. As the table illustrates, sensitivity asmeasured by detectable levels of a fluorescent dye improves as ball lenssize increases by a factor of 15 in moving from a 3 mm ball lens to a 10mm ball lens. TABLE 4 Different Size Ball Lens Experiment DescriptionGlass Bottom Plate with Solution Standards Cermax 300 w Xenon Lamp, dualexcitation and emission filtering Relative Sensitivity Sapphire BallLens with HEBBAR coating Molar equivalents of dye PMT - 3 mmCoAX - 10mmHB-DF 1.125E-12 PMT - 3 mmCoAX - 5 mmHB-DF 1.108E-11 PMT - 3 mmCoAX -3 mmHB-DF 1.727E-11

[0089] Protocols, Materials and Methods for the Experiments Herein

[0090] MDL1, minimum detectable level (MDL), is determined from thevariance of readings from many buffer blanks and would be affected bywell to well variability, positioning artifacts and other errors.

[0091] MDL2 is determined from the variance of repeated measurements ofthe same buffer blank and presumably would be affected only by the noiseof the detector.

[0092] The fiber optic assemblies were composed of fused silica coatedwith a black polyimide coating obtained from Fiberguide. The individualfibers are 200/220/240 in microns in diameter for thecore/cladding/coating respectively unless otherwise specifiied inparticular experiments.

[0093] The optical detectors utilized to evaluate fluorescent intensityin the experiments were either a Hamamatsu PMT and associatedelectronics as described in the Fluorocount instrument or a Hamamatsu HC135-01/100 Mhz PMT sensor module with embedded micro controller andRS-232-C interface. This sensor operates in the 360-650 nm range. ALabview™ software interface was written to control the PMT and acquiredata. When needed, excitation radiant power was measured using a NewportCorporation 1835-C power meter equipped with a 818-UV NIST traceablesilicon photodiode detector. The filters used in these experiments wereobtained from Chroma Technology Corporation or Omega Optical Inc., withthe exception of neutral density filters that were obtained from OrielCorp. In general and except where noted, all experiments were conductedwith the Hamamatsu PMT were double filtered on the excitation andemission ends with a 0.2 neutral density filter sandwiched in betweenthe interference filters. The excitation filters were IIQ475/40 +0.2ND+D480/20x. The emission filters were 535DF35t +0.2ND+535DF.

[0094] Three different light supplies were utilized for the experimentsand are identified as appropriate in the experimental results section.The first was a Quartz Tungsten Halogen (QTH) light obtained fromCole-Palmer Model #H-41700-00. The second was a Cermax LX-300W xenon Arcwith integral parabolic reflector. The third was a 175 watt Xenon Arclamp with ultra stable power supply from Hamamatsu.

[0095] All of the ball lenses were coated with HEBBAR. Experiments withthe Hamamatsu PMT were performed on a Newport Corporation optical benchwith Vibration dampening. Certain fixtures and mounts were speciallymade through local machine shops and others were obtained throughNewport Corporation.

[0096] Three types of plates were utilized. The standard plate is a 96well black top clear bottom polystyrene plate filled with fluorescentstandards. The glass bottom plates were specially modified blackpolystyrene 96 well plates with 175 micron glass bottoms. 384 well blackpolystyrene glass bottom plates were utilized for the 384 well readings.These specially modified plates were obtained frompolyfiltronics/Whitman.

Example 2 Sensitivity and Background Testing of Optical Assemblies ofOne Embodiment of the Invention

[0097] This example demonstrates the ability of the optical assembliesto achieve uniform illumination of the addressable wells while at thesame time avoiding illumination of the sides of the well and theillumination of adjacent wells. This leads to reduced backgroundfluorescent signals caused by reflections from the plate and wells andreduces punch through of excitation light through emission filters intodetection system, yet enables high sensitivity detection at twowavelengths.

[0098] This is exemplified by the determinations of minimumdetectability of a number of fluorescent standards. For example, theminimum detectable fluorescein level achieved using a detectorincorporating the optical system of the invention was better than 50 pMfluorescein in a standard 96 well plate. Emission was collected atwavelengths centered at both 535 nm and 580 nm. Both a blank solutionand a solution containing 2 nM fluorescein were measured. The minimumdetectable level (MDL) was calculated by generating a fluoresceincalibration curve that enabled the concentration of fluorescein that wasequivalent to 4 times the standard deviation of the buffer blank to becalculated. Because the detector typically measures changes ofbrightness within a single well, the standard deviations for readingswithin the same well at 1 Hz for eight seconds are given. It was foundthat the plate material also affected the MDL levels. Both buffer andfluorescein statistics were determined from 100 μL volumes in 40 wells(5 columns of 8 wells) of a 96 well plate. TABLE 5 Fluorescein MDLlevels measured using 480 ± 10 nm excitation 535 ± 17.5 nm and 580 ± 30nm emission filters. Plate bottom material Glass Glass PolystyrenePolystyrene Emission wavelength 535 nm 580 nm 535 nm 580 nm MDL (nMfluorescein) 0.0017 0.0085 0.034 0.072

[0099] Because the fluorescent dyes typically used with the detector arenot excited at wavelengths, more relevant standards are the fluorophores3-glycine chloro-coumarin (3GCC) and rhodamine 101. MDL measurementswere determined for these fluorescent dyes as described above exceptthat a fluorescent dye solution also containing 25 nM fluorophores3-glycine chloro-coumarin and 4 μM rhodamine 101 was used in place ofthe fluorescein solution TABLE 6 Two Dye MDL levels measured bothexcited using a 400 ± 7.5 nm filter. The 3GCC fluorescence was collectedusing a 460 ± 22.5 nm filter; the rhodamine 101 fluorescence wascollected using a 580 ± 30 nm filter. Fluorescent dye 3GCC rhodamine 101Plate bottom material Polystyrene polystyrene Emission wavelength 460 nm580 nm Excitation Wavelength 400 nm 400 nm MDL (nM fluorescent dye)0.181 20.8

[0100] Because 400 nm excitation light is not optimal for the efficientexcitation of rhodamine 101, the MDL level for this fluorophore isrelatively high when compared to those for 3GCC or fluorescein.

[0101] A desirable feature of the invention is that the fiber opticbundle and ball lens assemblies enable efficient excitation of theaddressable wells, as well as the ability to simultaneously measure atleast two optical properties. The average measured excitation intensityat 400 nm emerging through each of the fiber optic bundles and ball lensof the invention is 529±75 μW when using two 400±7.5 nm excitationfilters. The light source used was an ILC CXP300 300 watt Xenon arclamp, with 6.3 mm anti reflection coated fused silica ball lenses at thecommon ends of each of eight 5.18 mm diameter bundles containing 333fibers, 111 fibers from each leg of the randomly packed trifurcatedbundles. Light power was measured using a measured using a calibratedNewport 1835-C powermeter.

[0102] The use of the trifurcated fibers and ball lens system, and thecalculation of an emission ratio significantly reduces experimentalnoise, eliminates relative excitation variability between the 8 fiberoptic assemblies in the detector and leads to tighter C.V.s and improvesthe dynamic range of FRET based assays. A major additional advantage isthe removal of addition artifacts to enable continuous measurementsduring reagent addition. In these phenomena, intensities of cells loadedwith fluorescent dye often decline upon reagent addition. This declinein intensity may be due to some cells being washed from the detectionarea during addition and mixing of reagents. By taking the emissionratio at two separate wavelengths these artifacts are eliminated. In thedata set below, a mammalian neuronal cell line was loaded using a FRETbased fluorescent dye system. In this example, the majority of theemission change was in the 460 nm channel. For this experimentminelayers of mammalian cells were plated into the first 6 columns of a96 well plate. The emission intensities measurements were made at twowavelengths and the ratio determined for 35 seconds at 1 Hz for each ofthe 8 wells in a column. Reagent solutions were added following the12^(th) read of each column. In this example, test cells stimulated bydepolarization by addition of 100 μL high potassium solution (90 mM K).Control cells received normal Hank's buffered saline solution (HBS)without high potassium to test for addition artifacts. Both intensitydata and emission data were normalized versus basal levels to accountfor well to well variations in cell number or loading brightness andnormalized basal levels prior to reagent addition. This enables directcomparisons between intensity data and ratiometric data. TABLE 7Comparison of ratio versus non-ratio measurements Data Normalized toInitial Values Int (460 nm) Emission Ratio (460/580) HBS AV 91.7% 99.2%SD 4.0% 1.4% CV 4.4% 1.4% HiK AV 139.3% 155.6% SD 6.3% 4.9% CV 4.5% 3.1%Difference 47.6% 56.5%

[0103] As can be seen in table x, both the standard deviations andcoefficient of variation are about 30% lower for the ratiometric data(1.4% compared to 4.4% for HBS controls). There is also an additionartifact (91.7% of basal) in the intensity data but not in the emissiondata (99.2% of basal) for the control HBS additions. Because theemission ratio data factors both the increase in intensity at 460 nm andthe slight decrease in intensity at 580 nm upon depolarization with HiKsolution, the dynamic range of the emission ratio data is larger thanthat of the single intensity data. Statistics were determined from 24wells (3 rows of 8 wells).

Example 3 Determination of Na+ Dependent Depolarization in MammalianCells

[0104] An advantage of the use of the optical assemblies of theinvention is the ability to rapidly measure two wavelengthssimultaneously thereby enabling the rapid analysis of cellularresponses. In the field of voltage sensing, the use of rapiddepolarization measurements has several significant advantages overearlier relatively slow depolarization approaches that are subject toartifacts and reduce throughput of the assay. The use of the device thusallows the development of sensitive and rapid assay systems for membranevoltage measurements in whole cells. These assays are highly sensitive,reliable and able to discriminate relatively small changes in membranepotential with high precision.

[0105] Mammalian neuronal cells were grown in F12 complete mediumsupplemented with 20% fetal bovine serum. Prior to experiments cellswere washed twice with sodium free buffer (140 mM N-methyl-D-glucamine,10 mM HEPES, pH 7.2, 0.34 mM Na₂HPO₄, 0.4 mM MgCl₂, 0.5 mM KH₂PO₄, 5.37mM KCl, 1.26 mM CaCl₂, 2 g/L D-glucose). The cells were then harvestedusing calcium and magnesium free buffer and washed once. The cells werethen loaded with the fluorescent dye CC1-DMPE (4 μM for 30 minutes atroom temperature) and washed in sodium free buffer. The fluorescent dyeDiSBAC₂ was then added to the cells, after 30 minutes the plates wereloaded onto the device of the invention. All wells treated with achannel opener to open Na⁺ channels and maintained in low Na⁺ solution.Each well contained approximately 10⁵ cells. The average, standarddeviation, and standard error of the mean are given in the Table 8.TABLE 8 0 Na HBS HBS-TTX AV 99.5% 130.2% 98.9% SD 0.9% 4.3% 0.9% C.V.0.9% 3.3% 0.9% Difference N/A 30.7% −0.6%

Example 4 Determination of Dose Response Relationships

[0106] The large ratio changes observed with this method enable thecreation of highly reproducible assays and provide signals large enoughfor dose response curves to be generated. Furthermore because the devicecan acquire data continuously, the responses from the individual wellscan be viewed as a function of time. FIG. 8A shows the real time changesin voltage for individual wells.

[0107] The cells were stained and handled as described in Table 8. Allwells contained a sodium channel agonist . Traces show the effect ofdifferent doses of an anesthetic RS-105914-197 on blocking Na⁺ channelactivity in the neuronal cells. FIG. 8B shows the dose response of theanesthetic RS-105914-197 for blocking sodium channel activity using thedevice of the invention. The data represents the average of 4 wells andthe error bars represent the CV value. 1 mM of the drug completelyblocks the Na⁺ induced depolarization. These results with error analysisare summarized in Table 2. TABLE 2 Mean S.D. C.V.   0 mM RS-105914-197186.4% 3.7% 2.0% 0.1 mM RS-105914-197 172.2% 8.2% 4.7% 0.3 mMRS-105914-197 117.7% 5.6% 4.8% 1.0 mM RS-105914-197 100.5% 2.6% 2.6%

Example 5 Screening for Antagonists

[0108] To test whether it would be possible to identify antagonists on asingle plate assay in a screening format, a protocol was set up. Thisprotocol was designed such that compound additions were made from achemical multiwell plate to the test plate, and the wells readcontinuously during compound addition FIG. 9 demonstrates the use of thedevice to identify antagonists in a screening mode. The results showratio vs well number for the assay run in antagonist screening mode. Endratio values were averaged as in FIG. 9. A test antagonist (100 μM) wasused to test screening sensitivity. Vehicle control wells had anequivalent final concentration of DMSO as the test antagonist treatedwells. Negative controls received an addition of buffer instead ofagonist. In this experiment, cells (HEK-293) were washed with assaybuffer (160 mM NaCl, 10 mM HEPES, pH 7.4, 0.34 mM Na₂HPO₄, 0.4 mM MgCl₂,0.5 mM KH₂PO₄, 5.37 mM KCl, 1.26 mM CaCl₂, 2 g/L D-glucose) and loadedwith the fluorescent dyes CC2-DMPE and DiSBAC₂ as described in FIG. 7.

[0109] Publications

[0110] All publications, including patent documents and scientificarticles, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference.

[0111] All headings are for the convenience of the reader and should notbe used to limit the meaning of the text that follows the heading,unless so specified.

We claim:
 1. A method of simultaneously measuring at least two opticalproperties of emitted light from at least one sample in a plurality ofaddressable wells of a multiwell plate comprising the steps of, iv)aligning a plurality of addressable wells of a multiwell plate to aplurality of ball lenses; v) directing electromagnetic radiationsubstantially coaxially through the symmetry axis of each of saidplurality of ball lenses, vi) detecting the emitted light focused bysaid plurality of ball lenses from said at least one sample.
 2. Themethod of claim 1, wherein said electromagnetic radiation is directed tosaid plurality of ball lenses by at least one laser.
 3. The method ofclaim 1, wherein said electromagnetic radiation is directed to saidplurality of ball lenses by at least one fiber optic bundle.
 4. Themethod of claim 1, wherein said emitted light focused by said pluralityof ball lenses is directed to at least one detector through at least onefiber optic bundle.
 5. A device, comprising: i) a liquid handlercomprising at least one pipetting tip, said at least one pipetting tipcomprising programmable control of aspiration from a first plurality ofaddressable wells of a first multiwell plate and programmable control ofdispensation into a second plurality of addressable wells of a secondmultiwell plate, ii) an optical detector module comprising at least onedetector, said at least one detector being in optical connection to saidsecond plurality of addressable wells of said second multiwell plateand, said detector module simultaneously detecting at least two opticalproperties from each well of said second plurality of addressable wellsof said second multiwell plate, iii) a programmable moving conveyingsurface to align said first multiwell plate and said second multiwellplate to said liquid handler, and said detector module, and to move saidfirst multiwell plate and said second multiwell plate into and out ofsaid device, iv) a data processing and control module for coordinatingthe operation of said automatic measuring device, wherein said dataprocessing and control module coordinates said programmable movingconveying surface to move said second plurality of addressable wells ofsaid second multiwell plate to said liquid handler, wherein said liquidhandler simultaneously dispenses into said second addressable wells ofsaid second multiwell plate and said detector module simultaneouslymeasures at least two optical properties from each well of said secondplurality of addressable wells of said second multiwell plate, andwherein, said data processing and control module collects data from saiddetector module.
 6. The device of claim 5, wherein said data processingand control module intermittently collects data from said detectormodule.
 7. The device of claim 5, wherein said liquid handler dispensesinto said second addressable wells of said second multiwell plate andthere is a predefined delay before said detector module simultaneouslydetects at least two optical properties from each well of a plurality ofsaid second plurality of addressable wells of said second multiwellplate.
 8. The device of claim 5, wherein the optical property is lightemission at a particular wavelength.
 9. The device of claim 5, furthercomprising a optical irradiation module comprising at least one lightsource, wherein said at least one light source irradiates said secondplurality of addressable wells of said multiwell plate.
 10. The deviceof claim 5, wherein said at least one light source irradiates saidsecond plurality of addressable wells of said multiwell plateintermittently.
 11. The device of claim 5, wherein, said at least onelight source is programably controlled to irradiate said secondplurality of addressable wells of said multiwell plate at predefinedtimes.
 12. The device of claim 5, wherein said detector module furthercomprises at least one fiber optic bundle.
 13. The device of claim 8,wherein said at least one fiber optic bundle comprises at least onetrifurcated fiber optic bundle.
 14. The device of claim 5, wherein saiddetector module further comprises at least one ball lens.
 15. The deviceof claim 10, wherein said at least one ball lens is formed of a materialselected from the group consisting of glass, fused silica, quartz andsapphire.
 16. The device of claim 11, wherein said at least one balllens further comprises an anti-reflective coating.
 17. The device ofclaim 10, wherein said detector module further comprises at least onetrifurcated fiber optic bundle, said at least one trifurcated fiberoptic bundle having a diameter that is one third the diameter of said atleast one ball lens.
 18. The device of claim 10, wherein said detectormodule further comprises at least one trifurcated fiber optic bundle,said at least one trifurcated fiber optic bundle comprising at least onecentral optical fiber that is in direct optical communication with saidat least one light source, and wherein said at least one central fiberoptic bundle is coaxially aligned to said at least one ball lens.
 19. Adevice, comprising: i) an optical detector module comprising at leastone detector, said at least one detector being in optical connection toa plurality of addressable wells of a multiwell plate and, said detectormodule simultaneously detecting at any instant, at least two opticalproperties from each well of said plurality of addressable wells of saidmultiwell plate, ii) a optical irradiation module comprising at leastone light source, wherein said at least one light source irradiates saidplurality of addressable wells of said multiwell plate, iii) aprogrammable moving conveying surface to align said plurality ofaddressable wells of said multiwell plate to said detector module, andto move said plurality of addressable wells of said multiwell plate intoand out of said device, iv) an integration and control module forcoordinating the operation of said automatic measuring device, whereinsaid data processing and control module coordinates said programmablemoving conveying surface to move said plurality of addressable wells ofsaid multiwell plate to said detector module, wherein said detectormodule simultaneously detects at least two optical properties from eachwell of said second plurality of addressable wells of said secondmultiwell plate and, wherein said data processing and control modulecollects data from said detector module.
 20. The device of claim 19,wherein said data processing and control module intermittently collectsdata from said detector module.
 21. The device of claim 19, wherein theoptical property is light emission at a particular wavelength.
 22. Thedevice of claim 19, wherein said at least one light source irradiatessaid plurality of addressable wells of said multiwell plateintermittently.
 23. The device of claim 19, wherein, said at least onelight source is programably controlled to irradiate said plurality ofaddressable wells of said multiwell plate at predefined times.
 24. Thedevice of claim 19, wherein said detector module further comprises atleast one fiber optic bundle.
 25. The device of claim 24, wherein saidat least fiber optic bundle comprises a trifurcated fiber optic bundle.26. The device of claim 19, wherein said detector module furthercomprises at least one ball lens.
 27. The device of claim 26, whereinsaid at least one ball lens is formed of a material selected from thegroup consisting of glass, fused silica, quartz and sapphire.
 28. Thedevice of claim 27, wherein said at least one ball lens furthercomprises an anti-reflective coating.
 29. The device of claim 25,wherein said detector module further comprises at least one trifurcatedfiber optic bundle, said at least one trifurcated fiber optic bundlehaving a diameter that is one third the diameter of said at least oneball lens.
 30. The device of claim 25, wherein said detector modulefurther comprises at least one trifurcated fiber optic bundle, said atleast one trifurcated fiber optic bundle comprising a central opticalfiber bundle that is in direct optical communication with said at leastone light source, and wherein said central fiber optic bundle iscoaxially aligned to said at least one ball lens.
 31. The device ofclaim 25 wherein said at least one ball lens is about three times largerin diameter than said addressable wells of said multiwell plate.
 32. Thedevice of claim 25 wherein said at least one ball lens is about the samediameter as said addressable wells of said multiwell plate.
 33. Anoptical assembly, comprising a ball lens and a trifurcated fiber adaptedfor dual optical interrogation and in optical communication with saidball lens.
 34. The optical assembly of claim 33, wherein saidtrifurcated fiber comprises a first optically isolated emission bundleto collect light, second optically isolated emission bundle to collectlight, and an excitation bundle.
 35. The optical assembly of claim 34,wherein said ball lens is separated from said trifurcated fiber by atransmission space.
 36. The optical assembly of claim 35, wherein saidball lens comprises a sapphire material.
 37. The optical assembly ofclaim 36, wherein said ball lens comprises an anti-reflective coating.38. The optical assembly of claim 33, wherein said trifurcated fibercomprises a first plurality of emission bundles for receiving light of afirst wavelength and second plurality of emission bundles for receivinglight of a second wavelength and said first plurality of emissionbundles and said second plurality of emission bundles are randomlydistributed in plurality of excitation bundles.
 39. The optical assemblyof claim 33, wherein said trifurcated fiber comprises a first set ofbundles for transmitting light of a first wavelength and second set ofbundles for transmitting light of a second wavelength and third set ofbundles for transmitting light of a third wavelength.
 40. The opticalassembly of claim 39, wherein said trifurcated fiber is separated fromsaid ball lens by a transmission space of about 0.1 mm to 1 mm.
 41. Theoptical assembly of claim 35, wherein said ball lens comprises eithersapphire material or a silica material.
 42. The optical assembly ofclaim 39, wherein said first set of bundles and said second set ofbundles are coaxially arranged with respect to said third set ofbundles.
 43. An optical detection system, comprising: a) a light sourcethat launches at least one predetermined wavelength of light, b) sampleholder, c) a ball lens at a predetermined interrogation distance fromsaid sample holder, d) a trifurcated fiber adapted for dual opticalinterrogation and in optical communication with said ball lens, and e) adetector that detects light of at least one desired wavelength and inoptical communication with said ball lens.
 44. The optical detectionsystem of claim 43, wherein said trifurcated fiber comprises a firstplurality of emission bundles for receiving light of a first wavelengthand second plurality of emission bundles for receiving light of a secondwavelength and said first plurality of emission bundles and said lightsource launches at least one predetermined wavelength of excitationlight at said sample holder.
 45. The optical detection system of claim43, wherein said ball lens is at a predetermined transmission distancefrom said trifurcated fiber and further comprising at least onepositioner to controllably change said predetermined transmissiondistance.
 46. The optical detection system of claim 43, furthercomprising at least one positioner to controllably change saidpredetermined interrogation distance.
 47. The optical detection systemof claim 43, further comprising a liquid handling unit to dispenseliquids into addressable wells.
 48. The optical detection system ofclaim 43, further comprising a light activation system for triggeringliquid handling unit to dispense liquids into addressable wells.
 49. Theoptical detection system of claim 43, wherein said light source launcheslight through said trifurcated fiber to the location at least oneaddressable well in a sample in said sample holder to monitorepifluorescence.
 50. The optical detection system of claim 49, furthercomprising a computer control system to manage interrogation of asample.
 51. The optical detection system of claim 49, further comprisinga sample transfer system to transfer at least one sample platform tosaid sample holder.
 52. The optical detection system of claim 49,wherein said sample holder further comprises a positioning system. 53.The optical detection system of claim 43, wherein said ball lens is at apredetermined transmission distance from said trifurcated fiber thatapproximately corresponds to a focal length.
 54. The optical detectionsystem of claim 43, wherein said trifurcated fiber comprises an end andsaid end is generally at a focal plane of said ball lens.
 55. Theoptical detection system of claim 43, wherein an object to beinterrogated is generally at a focal plane of said ball lens.
 56. Anoptical fiber assembly, comprising a trifurcated fiber comprising afirst plurality of emission bundles for receiving light of a firstwavelength and second plurality of emission bundles for receiving lightof a second wavelength and said first plurality of emission bundles andsaid second plurality of emission bundles are non-randomly distributedin plurality of excitation bundles.
 57. The optical fiber assembly ofclaim 56, wherein said first set of bundles and said second set ofbundles are coaxially arranged with respect to said third set ofbundles.
 58. The optical fiber assembly of claim 56, wherein said firstset of bundles is coaxially arranged with respect to said second set ofbundles.
 59. A method of identifying a useful chemical, comprisinginterrogating a sample comprising a chemical of interest with one of theabove claimed devices, and detecting the activity of said chemical fromoptical signals from said device.
 60. A method of development for atherapeutic, comprising interrogating a sample comprising a chemicalwith of interest one of the above claimed devices, detecting theactivity of said chemical from optical signals from said device,administering said chemical or a chemical derived from the structure oractivity of said chemical to a cell, invertebrate or mammal, assessing atherapeutic effect of said administering and optionally adding asuitable pharmaceutical carrier to said chemical for administration intoa vertebrate or human.
 61. A chemical identified by a process ofdetecting optical signals from one of the above claimed devices, whereinsaid chemical is in a sample interrogated by said device.
 62. Apharmaceutical composition, comprising a pharmaceutical acceptablecarrier and a chemical identified by detecting optical signals from oneof the above claimed devices, wherein said chemical is in a sampleinterrogated by said device.